Apparatus and method for increasing insect resistance in transgenic plants

ABSTRACT

The present invention discloses a polynucleotide sequence for an insect salivary glucose oxidase enzyme and the amino acid sequence of the enzyme itself. It also provides recombinant polynucleotide vector systems designed to express the enzyme in a variety of host organisms. The invention also discloses a method for creating transgenic plants having increased resistance to insect predation resulting from the expression of the foreign glucose oxidase protein. The presence of the insect glucose oxidase enzyme triggers a plant&#39;s defensive mechanisms and results in increased resistance to insects.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/205,630, filed Dec. 3, 1998, the teachings of which are expressly incorporated herein by reference, which is a continuation-in-part of U.S. provisional patent application Ser. No. 60/067,457, filed Dec. 4, 1997, the teachings of which are expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a glucose oxidase enzyme isolated from the corn earworm Helicoverpa zea and the polynucleotide sequence that codes for it. More specifically, the invention. relates to recombinant polynucleotide vectors having a polynucleotide encoding a glucose oxidase enzyme isolated from an insect that may be inserted into a host cell or organism. The invention also relates to transgenic organisms having the polynucleotide sequence and expressing the polypeptide. Such vectors may be used to create transgenic plants that show increased resistance to a broad range of pathogens. The polynucleotide encoding the glucose oxidase may be inserted into a variety of commercial crop plants by means of a variety of recombinant vectors. The invention also relates to a recombinant antibody that may be used to detect the presence of the glucose oxidase enzyme

[0004] 2. Prior Art

[0005] The techniques of genetic engineering have been successfully applied to the pharmaceutical industry, resulting in a number of novel products. It has become apparent that the same technologies can be applied on a large scale to the production of enzymes of value to other industries. The benefits of achieving commercially useful processes through genetic engineering are expected to include cost savings in enzyme production, productions of enzymes in organisms generally recognized as safe which are suitable for food products, and specific genetic modifications at the genomic level to improve enzyme properties, such as thermal stability and performance characteristics, as well as those which would increase the ease with which the enzyme can be purified.

[0006] Genetic engineering and biotechnology are becoming increasingly important in the food industry. New technology may be used to make staple crops such as wheat, corn, potatoes and tomatoes easier and cheaper to grow. Transgenic plants, plants having genes from other organisms inserted into their cells, may be created that grow faster, grow in non-ideal environments or are more resistant to plant pathogens and insects. Transgenic plants that are sturdier or have strengthened defense mechanisms offer significant advantages to farmers around the world. In order to create transgenic plants, however, more must be understood about a plant's defenses against insects and other pathogens.

[0007] As will be appreciated by those skilled in the art, very little is understood about many of the complex interactions of insects and plants. For example, defoliation by soybean loopers triggers systemic acquired resistance to stem canker disease and redcrown rot. Conversly, stem-girdling by three-cornered alfalfa hoppers predisposes the same plants to the same diseases. Thus, the role of insects in triggering resistance or susceptibility to both insects and phytopathogens is still under investigation.

[0008] It has recently been recognized that the oral secretions from some herbivores trigger plants to release chemicals that attract the natural enemies of the herbivores feeding on the plants. For example, β-glucosidase in the regurgitant of Pieris brassicae caterpillars elicits the release of volatile compounds from cabbage leaves. More recently a glutamine-linolenic acid conjugate named volicitin was isolated from the regurgitant of beet armyworms Spodoptera exigua and found to induce the release of volatile chemicals from corn seedlings.

[0009] Monsanto has reported that natural plant defense responses to pathogen infection involve the production of active oxygen species including hydrogen peroxide (H₂O₂). Monsanto obtained transgenic potato plants expressing a fungal gene encoding glucose oxidase, which generates H₂O₂ when glucose is oxidized. H₂O₂ levels were elevated in both leaf and tuber tissues of these transgenic plants. Monsanto provided evidence that the trangenically elevated H₂O₂ levels enhanced disease resistance in potatoes against a broad range of plant pathogens. They report that the elevated levels of H₂O₂ in transgenic plants having a foreign glucose oxidase enzyme significantly increases the concentration of salicylic acid in leaf tissue, although no significant change was detected in the levels of free salicylic acid. The mRNAs of two defense-related genes encoding the anionic peroxidase and acidic chitinase were also induced. The results suggest that constitutively elevated sublethal levels of H₂O₂ are sufficient to activate an array of systemic host defense mechanisms, and these defense mechanisms may be a contributing factor to the H₂O₂-mediated disease resistance in transgenic plants. Systemic defense mechanisms, as opposed to local defense mechanisms, enhance resistance throughout the entire plant. Systemic defense mechanisms are therefore very desirable.

[0010] The transgenic potato tubers exhibited strong resistance to a bacterial soft rot disease caused by Erwinia carotovora and disease resistance was sustained under both aerobic and anaerobic conditions of bacterial infection. This resistance to soft rot was apparently mediated by elevated levels of H₂O₂ because the resistance could be counteracted by an exogenously added H₂O₂ degrading catalase.

[0011] The transgenic plants with increased levels of H₂O₂ also exhibited enhanced resistance to potato blight caused by Phytophthora infestans. The development of lesions resulting from infection by P. infestans was significantly delayed in leaves of these plants. Thus, the expression of active oxygen species-generating enzyme in transgenic plants represents a novel approach for engineering broad-spectrum, systemic disease resistance in plants.

[0012] The Salk Institute in La Jolla, Calif. has also used a similar approach to produce disease resistant rice. The Australian Science Foundation CSIRO scientists have developed a disease resistant cotton using the same gene. U.S. Pat. No.5,094,951 discusses the production of a fungal glucose oxidase in recombinant bacterial systems.

[0013] While glucose oxidase may be used with various agricultural applications, it may also be used for other applications. For example, glucose oxidase has been used with various food applications. U.S. Pat. No. 5,085,873 shows a process for the treatment of a non-food product for assuring its microbial decontamination. U.S. Pat. No. 4,996,062 shows a glucose oxidase food treatment and storage method. U.S. Pat. No. 4,990,343 shows an enzyme product and method of improving the properties of dough and the quality of bread. U.S. Pat. No. 4,957,749 shows a process for removing oxygen in foodstuffs and in drinks. U.S. Pat. No. 4,929,451 shows a process for eliminating disagreeable odor from soya milk. U.S. Pat. No. 4,557,927 shows various food products and processes for producing the same. U.S. Pat. No. 3,804,715 shows a process for preparing sugar containing maltose of high purity. U.S. Pat. No. 3,767,531 shows a preparation of insolubilized enzymes and U.S. Pat. No. 4,675,191 shows a method for production of a low alcoholic wine.

[0014] Glucose oxidase may also be used for other applications including biomedical and biochemical. For example, glucose oxidase may be used in the glucose monitoring of blood, urine, etc. as discussed by J. A. Lott and K. Turner in “Evaluation of Trinder's glucose oxidase method for measuring glucose in serum and urine,” Clin. Chem. 21 (12): 1745-1760 (1975). Another example is found in the product TES-TAPE® Lilly Glucose Enzymatic Test Strips. Yet another example is shown in U.S. Pat. No. 5,304,468, which shows a reagent test strip and apparatus for determination of blood glucose.

[0015] Glucose oxidase may also have other medical uses, such as the development of anticancer and/or antitumor agents as reported by C. F. Nathan and Z. A. Cohn in “Antitumor Effects of Hydrogen Peroxide in Vivo,” J. Exp. Med, Vol. 154, 1539-1553 (1981) and by Samoszuk M. D. Ehrlich and E. Ramzi in “Preclinical Safety Studies of Glucose Oxidase,” J. Pharmacol. Exp. Ther. 266(3):1643-1648 (1993). Also, as reported by P. Heiss, S. Bematz, G. Bruchelt and R. Senekowitsch-Schmidtke in “Cytotoxic Effect of Immunoconjugate Composed of Glucose Oxidase Coupled to a Chimeric Anti-ganglioside (GD2) Antibody on Spheroids,” Anticancer Res. 15(6A):2438-2439 (1995). They report that the therapeutic use of the chimeric anti-ganglioside (GD2) antibody shows some success in the therapy of neuroblastomas and melanoma as shown in various Phase I studies. To enhance the effect, glucose oxidase is coupled to the anti-GD2 antibody to produce H₂O₂ in the presence of glucose and oxygen. H₂O₂ easily penetrates the target cells in contrast to the antibody.

[0016] Glucose oxidase may also be used for the production of antimicrobial products such as soaps and cremes; for example, Kitchen Cupboard Almond Milk Kitchenhand Creme 2 oz. contains glucose and glucose oxidase. Also, glucose oxidase may be used in synthetic saliva, such as Biotene and the like, since many saliva contain an optimum concentration of a natural enzyme system that regulates the microbiological oral ecosystem (glucose oxidase+lactoperoxidase system).

[0017] Biochemical applications could also include Immunochemistry. For example, Vector offers VECTASTAIN® ABC kits of peroxidase, alkalinephosphatase and glucose oxidase for immunohistochemistry, ELISAS and blot detection. It could also be used for identifying and/or tracking proteins as reported by J. J. Marchalonis in “Enzymatic lodination of Proteins,” Biochemical Journal, 113, 229-305, (1969) and by J. I. Thorell and B. G. Johansson, Biochemica et Biophysica Acta, 251,363-9, (1969).

[0018] Other uses could include enzymatically amplified sensors for amperometry and voltammetry including electrodes designed for amperometric detection of glucose. For example, enzyme reactions have been widely explored in combination with the electrode chemical techniques to add specificity to voltammetry and amperometry. Such strategies are often referred to as “biosensors” since they employ a biomolecule (e g. enzyme, antibody) and can be used for sensing purposes. The most common situation is to use an oxidase enzyme to detect its primary substructrate (e.g. glucose oxidase to detect glucose). The enzyme typically oxidizes the substrate and then transfers reducing equivalence (electrons) to a small molecule (acceptor or mediator) which can be oxidized at the electrode surface. Electrodes designed for the amperometric detection of glucose, lactate and cholesterol are common examples which have used this technique.

[0019] Research has been conducted to design various different types of enzyme electrodes. Using the analyte molecule functioning as a mediator, a saturating excess of the enzyme's substrate is used to make the reduced enzyme kinetically inexhaustible. Once an analyte molecule is oxidized at an electrode surface, it is rapidly reduced by the enzyme and is hence available for re-oxidation. This means that each analyte molecule is detected several times on the experimental time scale, thus, the analytical signal is chemically amplified by the enzyme reaction. For example, catechol analytes using glucose oxidase have been proposed.

[0020] Thus, a need exists to utilize the interactions of insect enzymes with plants to improve agriculture. Specifically, there is a need to develop methods of increasing systemic plant resistance to pathogens using recombinant DNA technology. It is desirable to develop recombinant vectors encoding an insect glucose oxidase that may be introduced into a variety of plant species.

[0021] There is also a continuing demand for alternative sources of glucose oxidase for various fields including biomedical, biochemical, food production and preservation, and the like. It is therefore desirable to develop methods of manufacturing large quantities of stable glucose oxidase.

SUMMARY OF THE INVENTION

[0022] The present invention provides an isolated and purified DNA molecule comprising a DNA segment encoding a Helicoverpa zea, commonly known as the corn earworm, glucose oxidase (Gox) gene and methods for conferring enhanced resistance to insect predation by introducing and expressing this glucose oxidase gene in plant cells. The DNA molecule encoding Gox can encode an unaltered Gox or an altered Gox that substantially catalyzes the oxidation of glucose to form hydrogen peroxide. A DNA molecule of the invention may further comprise various known polynucleotide control sequences known to regulate the translation, transcription and function ofthe Gox gene. The polynucleotide sequence may also be encoded on an RNA molecule instead of DNA, as the nuleotide sequence is the significant aspect of the invention.

[0023] The present invention also discloses the amino acid sequence of the H. zea Gox protein that corresponds to the DNA sequence of the Gox Gene. As will be appreciated by those skilled in the art of biotechnology, this amino acid sequence may be altered by site-directed mutagenesis or enhanced by adding known amino acid sequences to either the N terminus or C terminus of the protein. For example, it is known that certain amino acid transit sequences direct a protein to specific areas within an organism. It is also well known in the art to add a polyhistidine sequence to a protein to facilitate purification. The addition of amino acid sequences to a known protein is usually accomplished by adding a DNA sequence upstream or downstream of the polynucleotide sequence that codes for the protein.

[0024] Insect salivary proteins may perform multiple physiological roles including induction or suppression of plant defense responses. The H. zea Gox was previously purified from salivary glands of the corn earworm and demonstrated to play an important role in inducing host resistance. The amino acid sequence of H. zea Gox is unique, sharing less than 30% homology with other reported Gox proteins. In particular, the novel protein structure shares very little homology with other proteins known to have the similar function of glucose oxidation. Because this protein has such a novel structure, it is unlikely that other glucose oxidases will confer the same type of resistance in plants. The corresponding Gox cDNA and two related genes from H. zea have been isolated. The Gox gene encodes a protein of 606 amino acids with an estimated molecular mass of 67 kD and a calculated isoelectric point of 5.26. The Gox gene is specifically expressed in the salivary gland of H. zea in a developmentally dependent manner. To determine the role of Gox in activation of host resistance, the Gox gene was introduced into tobacco plants via Agrobacterium-mediated transformation. Transgenic tobacco carrying the Gox transgene produces a large amount of Gox protein and exhibits high levels of Gox enzyme activity. Furthermore, the expression of Gox in transgenic tobacco was shown to significantly enhance host resistance against insect herbivory.

[0025] Expression of the H. zea Gox discourages predation of a transgenic plant by a variety of predators, including H. zea itself as well as other insects and pathogens. In addition, expression of Gox, even in relatively high sublethal concentrations, has no deleterious effects on the plant itself Plants grow at a normal rate into healthy plants. This makes the Gox gene especially well suited for creating transgenic commercial crops having increased resistance. Use of a recombinant Gox gene makes production of many crops, such as wheat, corn, soy beans, tomatoes and rice, both easier and cheaper.

[0026] The polynucleotide sequence encoding the Gox gene may also be used in microbial organisms, including bacteria and yeasts, to rapidly produce large quantities of Gox for other uses. Those skilled in the art of biotechnology will appreciate that various bacteria, such as E. coli, may be used as microbial factories to rapidly produce large amounts of a desired protein. It would be obvious to those skilled in the art that there are many ways to insert the disclosed Gox polynucleotide sequence into a bacteria and produce Gox on a large scale. H. zea Gox is a suitable protein for use in electrochemical sensing and analysis, measuring the level of sugar in blood or urine, voltammetry and any other application of a glucose oxidase.

[0027] Whether the Gox sequence is to be inserted into a transgenic plant to bolster its defenses to insect predation or placed in a microbe for large scale protein production, various known genetic sequences may be ligated both upstream and downstream from the Gox gene in order to regulate transcription and translation of the polynucleotide sequence. What additional nucleotide sequences are attached to the Gox sequence will depend on a wide variety of factors, such as the recombinant vector used, the type of cells into which the Gox gene is inserted, the method of inserting the genetic sequence, the desired level of expression of the Gox gene and other factors well known to those skilled in the art.

[0028] The present invention also discloses a recombinant antibody developed to ease detection of the protein. A significant portion of the Gox peptide sequence was used to develop an antibody that was shown to bind to the complete peptide and portions thereof Using the well known technique of an immunoblot assay, the presence of the protein may be readily detected.

[0029] It is therefore an object of the present invention to provide a polynucleotide sequence encoding an insect Gox protein that may be inserted into recombinant vectors that may be used to create transgenic plants having increased resistance to insect predation.

[0030] It is another object of the present invention to provide a polynucleotide sequence encoding an insect Gox protein that is suitable for large scale production in microbes of a glucose oxidase enzyme suitable for chemical, medical and industrial uses.

[0031] It is another object of the present invention to provide a polynucleotide sequence encoding an insect Gox protein that may be used in conjunction with know nucleotide sequences to enhance or modify translation and transcription of the Gox polynucleotide sequence.

[0032] It is another object of the present invention to provide recombinant vectors that may be used to insert a polynucleotide sequence coding for the Gox protein into a variety of organisms, including plants and microbes.

[0033] It is another object of the invention to provide antibodies that may be used to detect the presence of the Gox protein or a catalytically active portion thereof.

[0034] It is another object of the present invention to provide transgenic plants that express the Gox protein or a catalytically active portion thereof and exhibit enhanced resistance to insect predation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is a DNA blot illustrating the copy number of the Gox gene in H. zea genome. Total DNA was isolated from H. zea larvae by phenol/chloroform extraction. Ten microgram of genomic DNA was digested with EcoRI and HindIII, respectively, fractionated on a 1% agarose gel, and blotted onto a nylon membrane. The DNA blot was hybridized with a gene-specific probe (850˜900 bp of Gox) labeled with a PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals).

[0036]FIG. 2 are RNA blots illustrating the specific expression of the Gox gene in salivary glands during particular developmental stages. Total RNAs were isolated from the 6th instar H. zea at the different days using TRIzol reagent (Life Technology, Md.). Ten microgram of total RNA was separated on a 1.2% agarose gel containing formaldehyde and then transferred onto a nylon membrane. The RNA blots were hybridized with a gene-specific probe labeled with a PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals). The sample loading was verified by hybridizing with a labeled rDNA fragment from H. zea. A. RNA samples from salivary glands; B. RNA samples from whole larvae; C. RNA samples from the larvae from which salivary glands were removed.

[0037]FIG. 3 is an immunoblot illustrating the expression of the Gox protein in H. zea larvae. Total proteins were extracted from the 6th instar larvae. Twenty microgram of total protein extract was loaded onto each lane of 10% SDS-PAGE gel. After electrophoresis, separated proteins were transferred onto hybond-P PVDF membrane. The Gox protein was detected with the anti-Gox antibody using the ECL Plus detection system (Amersham).

[0038]FIG. 4 A. shows the transgenic tobacco carrying the 35S:Gox transgene. A. Morphologically normal transgenic plants; B. shows an immunoblot showing the overexpression of the Gox protein in transgenic plants as detected by immunoblot analysis with the anti-Gox antibody.

[0039]FIG. 5 is a graph showing the inhibitory effect of Gox expression in transgenic tobacco on larval growth of H. zea. Control: vector-transformed transgenics; Lox-Gox: transgenics with Gox levels less than 50 μmol/min/mg protein; high-Gox: transgenics with Gox levels higher than 100 μmol/min/mg protein.

[0040]FIG. 6 is a schematic diagram of a construct comprising the Gox gene and the PGEM-T Easy vector.

[0041]FIG. 7 is a schematic diagram of the pBK-CMV phagemid vector.

[0042]FIG. 8 is a schematic diagram of a construct comprising the BamH1/Sal1 portion of the Gox gene and the pET28a(+) vector.

[0043]FIG. 9 is a schematic diagram of a construct comprising Gox cDNA, the CaMV 35S promoter and the pCAMBIA vector.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The present invention provides a cDNA sequence encoding a glucose oxidase (Gox) from the corn earworm Helicoverpa zea. Knowledge of this sequence allows the expression in recombinant systems of polypeptides substantially similar to Gox, including Gox, analogs of Gox, and fragments of Gox.

[0045] The present invention also provides the amino acid sequences for which the Gox gene codes. Knowledge of these sequences allows the synthetic formation of these proteins, analogs of these proteins that perform the same catalytic function, and fragments of these proteins that induce the same enhanced insect resistance as the entire polypeptide.

[0046] In describing the present invention, the following terminology will be used in accordance with the definitions set out below. This terminology is well known to those skilled in the art.

[0047] “Glucose oxidase” or “Gox” refers to a polypeptide which catalyzes the oxidation of glucose to gluconic acid with the concomitant production of hydrogen peroxide. Procedures for determining glucose oxidase activity are known in the art.

[0048] “Recombinant polynucleotide” refers to a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature, and/or (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.

[0049] “Polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified, for example by methylation, phosphorylation, and/or by capping, and unmodified forms of the polynucleotide.

[0050] “Replicon” refers to any genetic element, e.g., a plasmid, a chromosome, a virus, that behaves as an autonomous unit of polynucleotide replication within a cell; i.e., capable of replication under its own control.

[0051] “Vector” is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment. Vectors may have one or more polynucleotide or recombinant polynucleotide and one or more control sequences.

[0052] “Control sequence” refers to polynucleotide sequences which are necessary to effect the expression and/or secretion of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and terminators; in eukaryotes, generally such control sequences include promoters, terminators and, in some instances enhancers. In addition, in both prokaryotes and eukaryotes, some control sequences direct the expressed polypeptide to a particular location within the cell or region within a multicellular organism. The term “control sequences” is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional polynucleotide sequences that influence the expression of a protein.

[0053] “Promoter” refers to a polynucleotide sequence upstream from an expressed polynucleotide. A promoter sequence signals the cellular machinery to express the polynucleotide downstream from it. Some promoters operate like a switch and only signal a cell to express a downstream polynucleotide under certain conditions, such as when the organism is under insect/pathogen attack, above a certain temperature, or in the presence of a particular chemical such as IPTG.

[0054] “Gene” is a polynucleotide sequence which is transcribed into mRNA and/or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the gene are determined by a translation start codon at the 5′-terminus and a translation stop codon at the 3′-terminus. A gene can include, but is not limited to, mRNA, cDNA, and recombinant polynucleotide sequences.

[0055] “Host cells”, “microbial cells”, “cells” and other terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities, are used interchangeably, and refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer polynucleotides, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to accidental or deliberate mutation. Progeny of the parental cell which are sufficiently similar to the parent can be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.

[0056] “Transformation” refers to the insertion of an exogenous polynucleotide into a microbial cell, or cells of a multcellular organism such as a plant, irrespective of the method used for insertion, for example, direct uptake, transduction, f-mating, particle bombardment or bacteria-mediated gene transfer. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0057] “Polypeptide” refers to the amino acid product of a sequence encoded within a polynucleotide, and does not refer to a specific length of the product, thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, sialylations, and the like.

[0058] IA. Molecular Cloning of Gox and Related Genes from H. zea

[0059]Helicoverpa zea eggs and neonates were obtained from the insect rearing facility at the Department of Entomology, University of Arkansas. Salivary glands were removed by dissection from the 6th instar of H. zea and stored at −80 C. The Poly(A) mRNAs were prepared from salivary glands of 6th instar H. zea larvae and used as templates for polymerase chain reaction (PCR) amplification. To isolate the Gox cDNA, two nested oligonucleotide primers (forward, 5′-ATGATYYTBGCYCARCARGA; 5′-CARACYG TBGTBGARG GHGC) were designed based on the N-terminal sequence (MILAQQDX GXQTVVEGASILNSXTAX VXTY) of Gox. The N-terminal sequence was obtained previously from purified Gox protein isolated from H. zea salivary glands. A reverse oligonucleotide primer (5′-CADCCDCCVARRMCYTTDCC) was designed according to a relatively conserved region (GKVLGGS) of Gox proteins. The amplified Gox fragments (about 340 bp) were purified from a 2% agarose gel and cloned into pGEM-T Easy plasmid vector (Promega).

[0060]FIG. 6 illustrates construct 50 which comprises Gox fragment 52 and pGEM-T Easy plasmid vector 54. The vector 54 is spliced open at restriction sites 60 and 58, using restriction enzymes. The Gox fragment 52 was then inserted into the open frame 56 in vector 54 using a ligase enzyme. Promoter 62 is located immediately upstream of fragment 52 and regulates expression of fragment 52. Sequence analysis indicated that the 340 bp cDNA fragment encodes a 114 amino acid peptide that matches the N-terminal sequence and conserved domains of Gox.

[0061] Although pGEM-T Easy vector was used in this particular embodiment, those skilled in the art will appreciate that there are several vectors that may be used when isolating a polynucleotide, and the techniques employed in creating constructs such as that shown in FIG. 6 are common. Vectors are available commercially and many researchers develop their own vectors to suit their preferences. Which vector is used will depend on the desired method of transformation, the organism to be transformed, size of the polynucleotide, personal preference, and other factors known to those skilled in the art.

[0062] To further isolate full-length Gox cDNAs, an H. zea cDNA library was constructed with mRNAs from salivary glands. The salivary gland-specific cDNA library has an average insert size of about 1.5 kb and a phage titer of 5×109 pfu/ml. ZAP Express phage vector (Stratagene, Calif.) was used to construct the library. Again, those skilled in the art will appreciate that the ZAP Express vector is only one of several vectors suitable for constructing a cDNA library.

[0063] The 340 bp segment isolated during the initial PCR amplification was purified from the pGEM-T Easy vector and radiolabeled with ³²P by random priming. Radiolabeling polynucleotides is an ubiquitous process used in molecular biology. Those skilled in the art will appreciate that this is only one of several suitable methods of radiolabeling and that ³²P is only one of several suitable isotopes.

[0064] Using the radiolabeled 340 bp Gox fragment as a probe, a full-length cDNA of the Gox gene was isolated by library screening and subsequent excision of phagemid pBK-CMV carrying ihe Gox cDNA from ZAP Express. FIG. 7 shows pBK-CMV plasmid 100 having the ColE1 origin 102, the lacZ control sequence 104, lac' control sequence 106, CMV control sequence 108 and Neomycin resistance control sequence 110. The Gox cDNA fragment is 1958 bp long (SEQ ID NO: 1)and encodes a protein of 606 amino acid residues (SEQ ID NO: 4). The Gox protein has an estimated molecular mass of 67 kD, with a calculated isoelectric point of 5.26. The N-terminal sequence encoded by this gene is identical with that of purified H. zea Gox enzyme, confirming that it indeed codes for the same Gox enzyme identified by the biochemical analysis. Southern blot analysis reveals that there are two copies of the Gox gene in the H. zea genome (FIG. 1).

[0065] In addition to the Gox genes, at least two additional H. zea genes, HZCE15 (SEQ ID. NO: 2) and HzCE20 (SEQ ID NO: 3) that share significant sequence homology with Gox have been identified. The HZCE15 cDNA (1842 bp) encodes a protein of 583 amino acids (SEQ ID NO: 5) with an estimated molecular mass of 64 kD and a calculated isoelectric point of 5.37. The HzCE20 cDNA (839 bp) encodes a protein of 214 amino acids (SEQ ID NO: 6) with an estimated molecular mass of 24 kD and a calculated isoelectric point of 5.35. These Gox proteins show very little homology, less than 30%, with all known glucose oxidases from other species.

[0066] IB. Salivary Gland-Specific Expression of Gox in H. zea.

[0067] To examine the tissue specificity and developmental variation of Gox expression, RNA blot analysis was carried out using the 6th instar H. zea. As shown in FIG. 2A, the Gox gene is specifically expressed at high levels in salivary glands. The Gox transcripts are also present in whole larvae (FIG. 2B), but they are hardly detectable in the larvae from which the salivary glands were removed (FIG. 2C). The expression of Gox is low at day 0 and 5 of the 6th instar, peaks at day 1 and 2, and decreases at day 3 and 4 (FIG. 2A and 2B).

[0068] The fact that Gox is found almost exclusively in H. zea salivary glands is a strong indication that the plant defense mechanisms induced by Gox evolved as a response to predation by the corn earworm.

[0069] IC. Anti-Gox Antibody and Immunoblot Analysis.

[0070] To obtain recombinant Gox antigen for antibody production, the BamHI/SalI fragment (from 232 to 917 bp) of the Gox cDNA was fused in frame to the BamHI/SalI site of pET28a(+) vector (Novagen). FIG. 8 illustrates construct 120 that comprises plasmid vector pET28a(+) 122 containing the BamHI1/SalI fragment 126. The vector 122 is spliced open at restriction sites 128 and 130, using restriction enzymes BarrHi and Sall respectively. The Gox fragment 126 was then inserted into the open frame 124 in vector 122 using a ligase enzyme. JPTG-inducible promoter 134 is located immediately upstream of the BamHI/SalI fragment 126 and regulates its expression. Control sequence 132 adds a polyhistidine sequence to fragment 126 to facilitate purification. Control sequence 136 confers kanamycin resistance to the plasmid 122. By adding kanamycin to the media in which the host E. coli cells are grown, only bacteria that were successfully transformed will survive. Those skilled in the art will appreciate that this technique of plasmid construction is well known and that a wide variety of vectors are just as suitable as pET28a(+).

[0071] Cells of E. coli BL21 ([)E3) strain transformed with the recombinant plasmid was grown at 37 C in Luria-Bertani medium containing 50 ug/ml kanamycin in order to select for only bacterial cells that were transformed. The polyhistidine-tagged Gox peptide was induced in mid-logarithmic bacterial cultures by addition of 1 mM IPTG. After 3 h growth, bacterial cells were harvested and the recombinant Gox protein was purified using B-PER 6× His Spin Purification Kit (Pierce). The purified protein was subsequently used for the production of rat anti-Gox antibody. Immunoblot analysis reveals that the anti-Gox antibody specifically detects the Gox protein in H. zea. During the stage of the 6th instar, the Gox protein level is very high at day 1, 2 and 3, and drastically decreases at day 4 (FIG. 3). These data are consistent with high Gox enzyme activities previously found in salivary glands during 1st, 2nd and 3rd days of the 6th instar (Eichenseer et al., 1999).

[0072] Glucose Oxidase Expression in Tobacco Transgenics

[0073] HIA. Generation of Transgenic Tobacco Expressing Glucose Oxidase

[0074] To produce transgenic tobacco with overexpression of Gox, the full-length Gox cDNA was placed under the control of a double CaMV 35S promoter and cloned into binary vector pCAMBIA 2300 (CAMBIA, Australia). FIG. 9 illustrates construct 140 which comprises Gox cDNA 142, inserted CaMV 35S promoter 144 and pCAMBIA2300 plasmid 148. Plasmid 148 was spliced open at restriction sites 154 and 156 using restriction enzymes. Promoter 144 and Gox cDNA 142 were then spliced together and inserted into open frame 152. The pCAMBIA plasmid contains an additional CaMV35S promoter 146 upstream of a Kanamycin resistance control sequence 158.

[0075] Those skilled in the art will appreciate that which vector and promoter are chosen for a particular construct depend on a variety of factors, including but not limited to the desired level of expression of the inserted gene, the plant into which the vector is to be inserted, and the method employed to insert the gene and create a transgenic plant. Other vectors suitable for introduction of the Gox gene into other organisms include but are not limited to pBI101 series, pBIN19, pGA482, and pRT-100 series.

[0076] The resulting construct and empty vector control were individually introduced into Agrobacterium tumafaciens strain EHA105. Twenty-six primary (T0) transgenic plants were generated via the Agrobacterium-mediated transformation. These transgenic plants exhibit normal growth and produce high levels of Gox protein (FIG. 6). The Gox transgene appears to encode two peptides with different molecular weights, which results from alternative translations. In addition to the expression of Gox protein, many transgenic plants show very high levels of Gox enzyme activity (Table 1). The Gox activity can be inherited from T0 plants to their progeny (T1 plants) as shown by five representative transgenic lines.

[0077] In this particular embodiment, Agrobacterium-mediated transformation was used to create transgenic plants. However, those skilled in the art will appreciate that a variety of transformation techniques are suitable for introducing a Gox polynucleotide sequence into a plant. These alternatives include, but are not limited to electroporation, microinjection, protoplast transformation, liposomal encapsulation, and microprojectile bombardment. TABLE 1 Glucose oxidase activity in selected transgenic lines (T0) and their progeny (T1). Transgenic Lines Gox activity (μmol/min/mg protein) G4-T0 189 G4-T1 (three progeny) 160, 87, 103 G11-T0 219 G11-T1 (three progeny) 50, 20, 35 G16-T0 129 G16-T1 (three progeny) 38, 19, 17 G24-T0 47 G24-T1 (three progeny) 15, 33, 22 G26-T0 63 G26-T1 (three progeny) 22, 19, 27

[0078] IIB. Transgenic Tobacco Exhibits Enhanced Insect Resistance.

[0079] To determine the effect of Gox expression in transgenic tobacco on insect resistance, feeding assays were conducted using two-day-old first instar H. zea larvae. Terminal leaves of transgenic tobacco plants were excised from 6-8 node stage plants grown in a greenhouse. Leaves were placed with moistened filter paper in 16 oz plastic deli tub containers. Six transgenic lines each were used for the vector-transformed control, low Gox (<50 μmol/min/mg protein) transgenics, and high Gox (>100 μmol/min/mg protein) transgenics. For each transgenic line, 20 larvae were placed in each container. After 4 days larvae were individually weighed to the nearest 0.1 mg.

[0080] As shown in FIG. 5, larvae grown on Gox transgenic plants showed significantly reduced weight gain compared to the wild type controls. Larval growth was more than 40% suppressed in the transgenic lines with the highest Gox activity (>100 μmol/min/mg protein). Our data demonstrate that expression of Gox in transgenic tobacco significantly enhance insect resistance.

[0081] In the present embodiment, a transgenic tobacco plant was created. However, those skilled in the art will appreciate that this same technique may be used on practically any other plant and will result in substantially the same enhanced insect resistance. Such other plants include, but are not limited to, wheat, rice, corn, potatoes, tomatoes, peas, soy beans, lettuce, melons, green beans, squash, and broccoli. Those skilled in the art will understand that this process would be suitable and advantageous for any commercially grown crop.

[0082] Promoters, a subclass of control sequences, are required in order for a polynucleotide to be expressed. There are many known promoters. Which promoter is best for a given transgenic organism will depend on the desired level of expression and the type of organism being transformed.

[0083] Those skilled in the art will appreciate that in addition to the wide variety of vectors available for the techniques described herein, there are also a wide variety of control sequences that may be added to a polynucleotide sequence for a variety of reasons. It is possible that in some or all plants the defensive response induced by Gox will be enhanced by directing the Gox protein to a specific location within the plant. This may be accomplished using control sequences that result in the addition of amino acids at either the N-terminus or C-terminus of the Gox protein. These added amino acids utilize mechanisms within a plant to direct the protein to which they are attached to specific regions of the plant cell. For example, some control sequences direct proteins to the chloroplasts. Some control sequences result in the protein attaching to a membrane. The techniques of utilizing theses control sequences to direct a certain protein to a certain location are well known to those skilled in the art.

[0084] It is also well known to those skilled in the art that control sequences may also be used to regulate both the translation and transcription of a polynucleotide sequence. These control sequences may be employed to regulate the concentration of the protein within the organism that is expressing it. The addition of these various types of control sequences to any given vector is a relatively simple procedure.

[0085] Some control sequences require the addition of a second, regulatory gene. For example, some control sequences inhibit gene translation only when an inhibitor protein is present. In this situation, it is necessary to add the gene that encodes the inhibitor protein to the vector. This inhibitor protein gene may in turn have its own control sequences upstream or downstream from it. It is even possible for an inhibitor protein gene to have a control sequence that requires a second inhibitor protein gene in order to function properly. However, this is generally not desirable because the more complex a system is, the more likely it is to fail. In addition, just as there are control sequences that, require inhibitor proteins, there are also control sequences that require activation proteins that increase gene translation. These control sequences require the addition of an activation protein gene.

[0086] There are also control sequences that regulate expression of coding sequences at the transcription stage. These sequences inhibit or facilitate ribosomal activity on mRNA. All of these, mechanisms are well known to those skilled in the art.

[0087] Which control sequences, promoters and plasmids are used for a particular plant will be depend on the method of transformation, the plant into which the vector is introduced and personal discretion. Another significant factor is the fact that hydrogen peroxide is harmful and even lethal to plants when present in large amounts. Greater expression of Gox leads to greater insect resistance. However, too high a level of Gox expression within a plant could lead to harmful amounts of hydrogen peroxide. Appropriate promoters and control sequences used in conjunction with a Gox polynucleotide sequence will induce expression of enough Gox to enhance insect resistance, but limit production of the Gox protein so that harmful levels of hydrogen peroxide are not produced. A secondary factor to consider when determining the appropriate amount of Gox expression will be the amount of glucose present in the environment into which the plant will be placed. This is due to the fact that increased concentrations of a substrate increases catalytic activity of an enzyme.

[0088] One method of insuring that Gox is expressed in acceptable amounts is to use a promoter that is induced by insect predation. Such a promoter assures that the Gox gene is only expressed when the plant has been attacked by feeding insects.

[0089] It may also be desirable to use only a portion of Gox gene or protein. Sometimes a protein is more effective for a particular purpose when only a fragment is used. This can be accomplished by inserting only a portion of the encoding polynucleotide sequence, or by using only a fragment of the protein itself Only a portion of the Gox protein is necessary to induce enhanced insect resistance. Similarly, only a portion of the Gox protein is necessary to catalyze the oxidation of glucose. In some situations it may be advantageous to use only the necessary portion of the polynucleotide or polypeptide rather than the entire sequence. This use of a protein fragment is well known to those skilled in the art.

[0090] Whereas, the present invention has been described in relation to the drawings attached hereto, it should be understood that other and further modifications, apart from those shown or suggested herein, may be made within the spirit and scope of this invention.

1 6 1 1958 DNA Helicoverpa zea CDS 15..1833 1 taggaaaata ccaag atg att ctg gcg cag caa gat tgc ggc tgc caa 48 Met Ile Leu Ala Gln Gln Asp Cys Gly Cys Gln 1 5 10 aca gta gta gaa ggt gcc agt atc ctg aac tcc aca gca tgt agc ggc 96 Thr Val Val Glu Gly Ala Ser Ile Leu Asn Ser Thr Ala Cys Ser Gly 15 20 25 acc tat ctg ttc atg gta ctc cta caa ggg tac tta tgg ggc cgc tgt 144 Thr Tyr Leu Phe Met Val Leu Leu Gln Gly Tyr Leu Trp Gly Arg Cys 30 35 40 gaa atc gcc acg cct tgc aag aga atc gaa tcc ata gat gaa aca gag 192 Glu Ile Ala Thr Pro Cys Lys Arg Ile Glu Ser Ile Asp Glu Thr Glu 45 50 55 tca gaa tat gac ttc ata gta gtg gga gct ggg tct tca gga tcc att 240 Ser Glu Tyr Asp Phe Ile Val Val Gly Ala Gly Ser Ser Gly Ser Ile 60 65 70 75 gta gct ggg aga ctg agc gaa aat act aca tat aaa gtc ctt cta tta 288 Val Ala Gly Arg Leu Ser Glu Asn Thr Thr Tyr Lys Val Leu Leu Leu 80 85 90 gaa gct ggt ggg ccc gaa cct ctg ggt gct cgt gtc cca tca ttt tac 336 Glu Ala Gly Gly Pro Glu Pro Leu Gly Ala Arg Val Pro Ser Phe Tyr 95 100 105 aaa act ttc tgg ggc cac gat gag gta gac tgg caa ggg cga gcc gta 384 Lys Thr Phe Trp Gly His Asp Glu Val Asp Trp Gln Gly Arg Ala Val 110 115 120 cct gat ccc aac ttc tgc cgc gac caa gga gaa ctc ggg tgc caa tgg 432 Pro Asp Pro Asn Phe Cys Arg Asp Gln Gly Glu Leu Gly Cys Gln Trp 125 130 135 ccg cta gga aaa agc tta gga gga tct agt ctc ctc aac ggt atg atg 480 Pro Leu Gly Lys Ser Leu Gly Gly Ser Ser Leu Leu Asn Gly Met Met 140 145 150 155 tac cac aaa ggc cac gcc gct gac tac gag acc tgg gtg gaa gaa ggc 528 Tyr His Lys Gly His Ala Ala Asp Tyr Glu Thr Trp Val Glu Glu Gly 160 165 170 gcg gaa ggt tgg tcc tgg gac gag gtc aaa cca ttc atg gac ttg gcc 576 Ala Glu Gly Trp Ser Trp Asp Glu Val Lys Pro Phe Met Asp Leu Ala 175 180 185 gaa ggt aac aga caa gtg gga agc ctc gtc gaa ggg aag tac cac tcc 624 Glu Gly Asn Arg Gln Val Gly Ser Leu Val Glu Gly Lys Tyr His Ser 190 195 200 gaa act gga cgc atg cca ata caa aca ttt aac tac cag ccc ccg cag 672 Glu Thr Gly Arg Met Pro Ile Gln Thr Phe Asn Tyr Gln Pro Pro Gln 205 210 215 ctt agg gat cta ata gaa gca atc aac cag acg gga ctg ccg atc atc 720 Leu Arg Asp Leu Ile Glu Ala Ile Asn Gln Thr Gly Leu Pro Ile Ile 220 225 230 235 acg gac atg aac aac ccg aac aca ccc gac ggc ttt gta gta gca caa 768 Thr Asp Met Asn Asn Pro Asn Thr Pro Asp Gly Phe Val Val Ala Gln 240 245 250 acc ttt aat gac aat ggc cag cgc tac acc aca gcc cgc gca tac ctg 816 Thr Phe Asn Asp Asn Gly Gln Arg Tyr Thr Thr Ala Arg Ala Tyr Leu 255 260 265 gct ccc aaa tcc gag cga ccc aac ctg agc gtc aaa ctg tac gcc cac 864 Ala Pro Lys Ser Glu Arg Pro Asn Leu Ser Val Lys Leu Tyr Ala His 270 275 280 gtg act aaa gta ctc ttc gac ggg aag aag gct gtg gga gtg gaa tac 912 Val Thr Lys Val Leu Phe Asp Gly Lys Lys Ala Val Gly Val Glu Tyr 285 290 295 gtc gac aag aac ggc aat acg aaa act gtc aag act act aaa gag gta 960 Val Asp Lys Asn Gly Asn Thr Lys Thr Val Lys Thr Thr Lys Glu Val 300 305 310 315 atc gtg tca gca gga cca ttg acc agt cct aaa atc ctg atg cat tct 1008 Ile Val Ser Ala Gly Pro Leu Thr Ser Pro Lys Ile Leu Met His Ser 320 325 330 ggt gtg gga cct aaa gag gta tta gag cca ttg ggc att ccc gta gta 1056 Gly Val Gly Pro Lys Glu Val Leu Glu Pro Leu Gly Ile Pro Val Val 335 340 345 gct gac gtg ccc gtc ggt aag agg ctg agg aac cac tgc ggg gcc acg 1104 Ala Asp Val Pro Val Gly Lys Arg Leu Arg Asn His Cys Gly Ala Thr 350 355 360 ctg aac ttc ctc ctg aag aag tcc aac aac acc cag tct ttg gac tgg 1152 Leu Asn Phe Leu Leu Lys Lys Ser Asn Asn Thr Gln Ser Leu Asp Trp 365 370 375 agt gca ctg act gac tac ttg ctg gaa ctt gac ggg cct atg agt tcc 1200 Ser Ala Leu Thr Asp Tyr Leu Leu Glu Leu Asp Gly Pro Met Ser Ser 380 385 390 395 act ggg ctt act cag ctc act ggc ctc cta tac tca agc tac gca gac 1248 Thr Gly Leu Thr Gln Leu Thr Gly Leu Leu Tyr Ser Ser Tyr Ala Asp 400 405 410 aag agt cgc aag cag cca gac cta cag ttc ttc ttc aac ggt ctg tat 1296 Lys Ser Arg Lys Gln Pro Asp Leu Gln Phe Phe Phe Asn Gly Leu Tyr 415 420 425 gct gac tgc tcc aag act ggt gtt atc ggc gaa ccg gct gag gac tgc 1344 Ala Asp Cys Ser Lys Thr Gly Val Ile Gly Glu Pro Ala Glu Asp Cys 430 435 440 agc gat ggc tac aaa atc tca gcg aat gcc gta gcc ctg ctc ccg cgc 1392 Ser Asp Gly Tyr Lys Ile Ser Ala Asn Ala Val Ala Leu Leu Pro Arg 445 450 455 agc gtg ggc cac gtg acc atc aac tcg aca gac ccc ttc aag tca gcg 1440 Ser Val Gly His Val Thr Ile Asn Ser Thr Asp Pro Phe Lys Ser Ala 460 465 470 475 ctg ttc tac ccc aac ttc ttc tct cac cca gac gac atg aac atc gtg 1488 Leu Phe Tyr Pro Asn Phe Phe Ser His Pro Asp Asp Met Asn Ile Val 480 485 490 atg gaa ggc gtt gat tac tta cgc aag att ttc gaa agt cag gtg cta 1536 Met Glu Gly Val Asp Tyr Leu Arg Lys Ile Phe Glu Ser Gln Val Leu 495 500 505 caa gag aaa tac aaa gta gaa cta gac ccg gaa tac aca caa gag tgt 1584 Gln Glu Lys Tyr Lys Val Glu Leu Asp Pro Glu Tyr Thr Gln Glu Cys 510 515 520 gac gac tac aaa gcg tgg tca aga gac tgg aag gag tgt atg att cgc 1632 Asp Asp Tyr Lys Ala Trp Ser Arg Asp Trp Lys Glu Cys Met Ile Arg 525 530 535 cta cac act gac cca cag aac cat cag ctc gcc acc aac gct att ggc 1680 Leu His Thr Asp Pro Gln Asn His Gln Leu Ala Thr Asn Ala Ile Gly 540 545 550 555 aag gtc gtt gac ccg cag ctt agg gtt tat gac gtc aaa aac ttg cga 1728 Lys Val Val Asp Pro Gln Leu Arg Val Tyr Asp Val Lys Asn Leu Arg 560 565 570 gta tgt gac gcg ggt tca atg ccc tca ccg ccg acc ggc aac cct caa 1776 Val Cys Asp Ala Gly Ser Met Pro Ser Pro Pro Thr Gly Asn Pro Gln 575 580 585 ggc gct atc atg gtg gta gca gaa cga tgc gca cac ttc atc aaa cag 1824 Gly Ala Ile Met Val Val Ala Glu Arg Cys Ala His Phe Ile Lys Gln 590 595 600 act tgg caa tagaaccacg agcttacaac atatacccta tgcaatgtta 1873 Thr Trp Gln 605 tccttgttat tatcaattac gtacataaag tacgagaatt atgttcctct 1923 tccagtgaat aaaatgttga atatttaaaa aaaaa 1958 2 1842 DNA Helicoverpa zea CDS 17..1765 2 gtcacatctc acagca atg gca gac gcg gct gct tcc cag tca tcg gcg 49 Met Ala Asp Ala Ala Ala Ser Gln Ser Ser Ala 1 5 10 cag acg gtc aag ctt gct ctg caa gtg ctg cag acg ctg agc ctc acc 97 Gln Thr Val Lys Leu Ala Leu Gln Val Leu Gln Thr Leu Ser Leu Thr 15 20 25 gcg tgg cag tac ccg cct gac tgc gct ctc act aat ggg agc tca ttc 145 Ala Trp Gln Tyr Pro Pro Asp Cys Ala Leu Thr Asn Gly Ser Ser Phe 30 35 40 gac ttc ata gtg gtg ggg agt ggc acc gct ggg tca gtg ctg gcc aac 193 Asp Phe Ile Val Val Gly Ser Gly Thr Ala Gly Ser Val Leu Ala Asn 45 50 55 agg ctg tca gcc aac gat agt gtc agt gtg ttg ctc ctt gaa gcg gga 241 Arg Leu Ser Ala Asn Asp Ser Val Ser Val Leu Leu Leu Glu Ala Gly 60 65 70 75 gga tat cca cca ttg gag tca gag ctg cct gcg ctt ttc atg atg tta 289 Gly Tyr Pro Pro Leu Glu Ser Glu Leu Pro Ala Leu Phe Met Met Leu 80 85 90 agc aac tcg gac tat gac tac aaa tac tac gca gaa aac gac aac tac 337 Ser Asn Ser Asp Tyr Asp Tyr Lys Tyr Tyr Ala Glu Asn Asp Asn Tyr 95 100 105 acg atg cag aac ata cga ggg aag aga tgt gcc ctc act caa ggc aag 385 Thr Met Gln Asn Ile Arg Gly Lys Arg Cys Ala Leu Thr Gln Gly Lys 110 115 120 gtg ttg gga ggc acc agc tcc acc tat gcc atg atg cat acg aga ggt 433 Val Leu Gly Gly Thr Ser Ser Thr Tyr Ala Met Met His Thr Arg Gly 125 130 135 gat cct cag gac tac gac gtg tgg gcg gag agg gcc aac gat aca acc 481 Asp Pro Gln Asp Tyr Asp Val Trp Ala Glu Arg Ala Asn Asp Thr Thr 140 145 150 155 tgg aat gcc acc aac aca ttg tca tat ttc aag aaa caa gaa aaa cta 529 Trp Asn Ala Thr Asn Thr Leu Ser Tyr Phe Lys Lys Gln Glu Lys Leu 160 165 170 act gac gaa gaa ctc ctg cac tct gag tac gct gct gtc cac ggt act 577 Thr Asp Glu Glu Leu Leu His Ser Glu Tyr Ala Ala Val His Gly Thr 175 180 185 gat ggg atg gtc aaa ata aga aga gag aca agt cct ctt ctc gat gat 625 Asp Gly Met Val Lys Ile Arg Arg Glu Thr Ser Pro Leu Leu Asp Asp 190 195 200 att ctt gga cgc att tta aga ggg cgg gca cga ttt aat gat gga cac 673 Ile Leu Gly Arg Ile Leu Arg Gly Arg Ala Arg Phe Asn Asp Gly His 205 210 215 cac atc att gaa agt ctt cgc ttc ggc tac aca cag gtt gct ata tgc 721 His Ile Ile Glu Ser Leu Arg Phe Gly Tyr Thr Gln Val Ala Ile Cys 220 225 230 235 cat cga atg atg gag tgc ggg caa agt agt gca ctt gcc tat cta agt 769 His Arg Met Met Glu Cys Gly Gln Ser Ser Ala Leu Ala Tyr Leu Ser 240 245 250 tct gcc aag aaa cga aag aat cta tgt gta tct tta ttt act act gct 817 Ser Ala Lys Lys Arg Lys Asn Leu Cys Val Ser Leu Phe Thr Thr Ala 255 260 265 acc aag atc ttg atc gag aat gag gtc gct gtt ggt gta cag ctg acg 865 Thr Lys Ile Leu Ile Glu Asn Glu Val Ala Val Gly Val Gln Leu Thr 270 275 280 acc tct acc aac gaa aca tac aat ata tat tcc aac aag gaa gtg att 913 Thr Ser Thr Asn Glu Thr Tyr Asn Ile Tyr Ser Asn Lys Glu Val Ile 285 290 295 gtg tct gct ggc acc ttc aac agt ccc aag cta ctg atg ctg tct ggc 961 Val Ser Ala Gly Thr Phe Asn Ser Pro Lys Leu Leu Met Leu Ser Gly 300 305 310 315 att gga ccc cgc gaa cat ttg gag tca gtc gaa atc gac gtg gtt gct 1009 Ile Gly Pro Arg Glu His Leu Glu Ser Val Glu Ile Asp Val Val Ala 320 325 330 gac ctt cca gtc ggc cag aac tac atg gat cag ccg agt gct cca att 1057 Asp Leu Pro Val Gly Gln Asn Tyr Met Asp Gln Pro Ser Ala Pro Ile 335 340 345 att atc caa atg gat gaa agt gca gaa gta gct ggt gcc ata aac cca 1105 Ile Ile Gln Met Asp Glu Ser Ala Glu Val Ala Gly Ala Ile Asn Pro 350 355 360 cac cag ttc cct ctg cca act ttc att ggg aac gta gcc ctc gat tca 1153 His Gln Phe Pro Leu Pro Thr Phe Ile Gly Asn Val Ala Leu Asp Ser 365 370 375 ccc agc aag cga cca caa tac cac acg gtc aat ttt ctg ttc ccg gcg 1201 Pro Ser Lys Arg Pro Gln Tyr His Thr Val Asn Phe Leu Phe Pro Ala 380 385 390 395 aac tcc aca gat cta ctg gat atg tgt tcc ttg ttt ctt agt tac tcg 1249 Asn Ser Thr Asp Leu Leu Asp Met Cys Ser Leu Phe Leu Ser Tyr Ser 400 405 410 gat gaa gtt tgc cag aag gtc tac gag gcg acc acg aac cga aag act 1297 Asp Glu Val Cys Gln Lys Val Tyr Glu Ala Thr Thr Asn Arg Lys Thr 415 420 425 ata ttt tcc ttg gta ggg ttg gcg tta cca aac tct aga ggt gaa gta 1345 Ile Phe Ser Leu Val Gly Leu Ala Leu Pro Asn Ser Arg Gly Glu Val 430 435 440 ttg ctg gcc agc gct gac cct gct gca gca cca atc gtt cac act ggc 1393 Leu Leu Ala Ser Ala Asp Pro Ala Ala Ala Pro Ile Val His Thr Gly 445 450 455 atg ttc agc aac tat acg gac ttg aac ctg atg ggg cgc gct ttt atc 1441 Met Phe Ser Asn Tyr Thr Asp Leu Asn Leu Met Gly Arg Ala Phe Ile 460 465 470 475 gac cac gta aga gtg ctg aac tct act tac ttc cga agc gta aac gcg 1489 Asp His Val Arg Val Leu Asn Ser Thr Tyr Phe Arg Ser Val Asn Ala 480 485 490 aca atc ttg gac ctt ggc ttc tgc aag gat aca aca agt gag gtg gaa 1537 Thr Ile Leu Asp Leu Gly Phe Cys Lys Asp Thr Thr Ser Glu Val Glu 495 500 505 ttc tgg gag tgc tac acg ttg gct atg tcg aat acc atg tgg cac ttt 1585 Phe Trp Glu Cys Tyr Thr Leu Ala Met Ser Asn Thr Met Trp His Phe 510 515 520 gga ggg act tgt gcc atg ggt ctg gtg ctg gac agc aag atg aag gtc 1633 Gly Gly Thr Cys Ala Met Gly Leu Val Leu Asp Ser Lys Met Lys Val 525 530 535 aaa ggc gtg ggg agg ctg agg gtg gtg gac tcc tcc tcc atg ccg gcg 1681 Lys Gly Val Gly Arg Leu Arg Val Val Asp Ser Ser Ser Met Pro Ala 540 545 550 555 ctt gtt acc ggg aag gtc aac tct ccg atc ggc atg ctt gct gaa aaa 1729 Leu Val Thr Gly Lys Val Asn Ser Pro Ile Gly Met Leu Ala Glu Lys 560 565 570 gct gct gat ttc ata cta act gat cat aac atc ttg taactctctt 1775 Ala Ala Asp Phe Ile Leu Thr Asp His Asn Ile Leu 575 580 gaaacaatta caatttggtc aataaaaatt aaatgagtta aaacgtgaaa aaaaaaaaaa 1835 aaaaaaa 1842 3 824 DNA Helicoverpa zea CDS 63..704 3 acaacaccca gtctttggac tggagtgcac tgactgacta cttgctggaa 50 cttgacgggc ct atg agt tcc act ggg ctt act cag ctc act ggc ctc 98 Met Ser Ser Thr Gly Leu Thr Gln Leu Thr Gly Leu 1 5 10 cta tac tca agc tac gca gac aag agt cgc aag cag cca gac cta cag 146 Leu Tyr Ser Ser Tyr Ala Asp Lys Ser Arg Lys Gln Pro Asp Leu Gln 15 20 25 ttc ttc ttc aac ggt ctg tat gct gac tgc tcc aag act ggt gtt atc 194 Phe Phe Phe Asn Gly Leu Tyr Ala Asp Cys Ser Lys Thr Gly Val Ile 30 35 40 ggc gaa ccg gct gag gac tgc agc gat ggc tac aaa atc tca gcg aat 242 Gly Glu Pro Ala Glu Asp Cys Ser Asp Gly Tyr Lys Ile Ser Ala Asn 45 50 55 60 gcc gta gcc ctg ctc ccg cgc agc gtg ggc cac gtg acc atc aac tcg 290 Ala Val Ala Leu Leu Pro Arg Ser Val Gly His Val Thr Ile Asn Ser 65 70 75 aca gac ccc ttc aag tca gcg ctg ttc tac ccc aac ttc ttc tct cac 338 Thr Asp Pro Phe Lys Ser Ala Leu Phe Tyr Pro Asn Phe Phe Ser His 80 85 90 cca gac gac atg aac atc gtg atg gaa ggc gtt gat tac tta cgc aag 386 Pro Asp Asp Met Asn Ile Val Met Glu Gly Val Asp Tyr Leu Arg Lys 95 100 105 att ttc gaa agt cag gtg cta caa gag aaa tac aaa gta gaa cta gac 434 Ile Phe Glu Ser Gln Val Leu Gln Glu Lys Tyr Lys Val Glu Leu Asp 110 115 120 ccg gaa tac aca caa gag tgt gac gac tac aaa gcg tgg tca aga gac 482 Pro Glu Tyr Thr Gln Glu Cys Asp Asp Tyr Lys Ala Trp Ser Arg Asp 125 130 135 140 tgg aag gag tgt atg att cgc cta cac act gac cca cag aac cat cag 530 Trp Lys Glu Cys Met Ile Arg Leu His Thr Asp Pro Gln Asn His Gln 145 150 155 ctc gcc acc aac gct att ggc aag gtc gtt gac ccg cag ctt agg gtt 578 Leu Ala Thr Asn Ala Ile Gly Lys Val Val Asp Pro Gln Leu Arg Val 160 165 170 tat gac gtc aaa aac ttg cga gta tgt gac gcg ggt tca atg ccc tca 626 Tyr Asp Val Lys Asn Leu Arg Val Cys Asp Ala Gly Ser Met Pro Ser 175 180 185 ccg ccg acc ggc aac cct caa ggc gct atc atg gtg gta gca gaa cga 674 Pro Pro Thr Gly Asn Pro Gln Gly Ala Ile Met Val Val Ala Glu Arg 190 195 200 tgc gca cac ttc atc aaa cag act tgg caa tagaaccacg agcttacaac 724 Cys Ala His Phe Ile Lys Gln Thr Trp Gln 205 210 atatacccta tgcaatgtta tccttgttat tatcaattac gtacataaag 774 tacgagaatt atgttcctct tccagtgaat aaaatgttga atatttaaaa 824 4 606 PRT Helicoverpa zea 4 Met Ile Leu Ala Gln Gln Asp Cys Gly Cys Gln Thr Val Val Glu Gly 1 5 10 15 Ala Ser Ile Leu Asn Ser Thr Ala Cys Ser Gly Thr Tyr Leu Phe Met 20 25 30 Val Leu Leu Gln Gly Tyr Leu Trp Gly Arg Cys Glu Ile Ala Thr Pro 35 40 45 Cys Lys Arg Ile Glu Ser Ile Asp Glu Thr Glu Ser Glu Tyr Asp Phe 50 55 60 Ile Val Val Gly Ala Gly Ser Ser Gly Ser Ile Val Ala Gly Arg Leu 65 70 75 80 Ser Glu Asn Thr Thr Tyr Lys Val Leu Leu Leu Glu Ala Gly Gly Pro 85 90 95 Glu Pro Leu Gly Ala Arg Val Pro Ser Phe Tyr Lys Thr Phe Trp Gly 100 105 110 His Asp Glu Val Asp Trp Gln Gly Arg Ala Val Pro Asp Pro Asn Phe 115 120 125 Cys Arg Asp Gln Gly Glu Leu Gly Cys Gln Trp Pro Leu Gly Lys Ser 130 135 140 Leu Gly Gly Ser Ser Leu Leu Asn Gly Met Met Tyr His Lys Gly His 145 150 155 160 Ala Ala Asp Tyr Glu Thr Trp Val Glu Glu Gly Ala Glu Gly Trp Ser 165 170 175 Trp Asp Glu Val Lys Pro Phe Met Asp Leu Ala Glu Gly Asn Arg Gln 180 185 190 Val Gly Ser Leu Val Glu Gly Lys Tyr His Ser Glu Thr Gly Arg Met 195 200 205 Pro Ile Gln Thr Phe Asn Tyr Gln Pro Pro Gln Leu Arg Asp Leu Ile 210 215 220 Glu Ala Ile Asn Gln Thr Gly Leu Pro Ile Ile Thr Asp Met Asn Asn 225 230 235 240 Pro Asn Thr Pro Asp Gly Phe Val Val Ala Gln Thr Phe Asn Asp Asn 245 250 255 Gly Gln Arg Tyr Thr Thr Ala Arg Ala Tyr Leu Ala Pro Lys Ser Glu 260 265 270 Arg Pro Asn Leu Ser Val Lys Leu Tyr Ala His Val Thr Lys Val Leu 275 280 285 Phe Asp Gly Lys Lys Ala Val Gly Val Glu Tyr Val Asp Lys Asn Gly 290 295 300 Asn Thr Lys Thr Val Lys Thr Thr Lys Glu Val Ile Val Ser Ala Gly 305 310 315 320 Pro Leu Thr Ser Pro Lys Ile Leu Met His Ser Gly Val Gly Pro Lys 325 330 335 Glu Val Leu Glu Pro Leu Gly Ile Pro Val Val Ala Asp Val Pro Val 340 345 350 Gly Lys Arg Leu Arg Asn His Cys Gly Ala Thr Leu Asn Phe Leu Leu 355 360 365 Lys Lys Ser Asn Asn Thr Gln Ser Leu Asp Trp Ser Ala Leu Thr Asp 370 375 380 Tyr Leu Leu Glu Leu Asp Gly Pro Met Ser Ser Thr Gly Leu Thr Gln 385 390 395 400 Leu Thr Gly Leu Leu Tyr Ser Ser Tyr Ala Asp Lys Ser Arg Lys Gln 405 410 415 Pro Asp Leu Gln Phe Phe Phe Asn Gly Leu Tyr Ala Asp Cys Ser Lys 420 425 430 Thr Gly Val Ile Gly Glu Pro Ala Glu Asp Cys Ser Asp Gly Tyr Lys 435 440 445 Ile Ser Ala Asn Ala Val Ala Leu Leu Pro Arg Ser Val Gly His Val 450 455 460 Thr Ile Asn Ser Thr Asp Pro Phe Lys Ser Ala Leu Phe Tyr Pro Asn 465 470 475 480 Phe Phe Ser His Pro Asp Asp Met Asn Ile Val Met Glu Gly Val Asp 485 490 495 Tyr Leu Arg Lys Ile Phe Glu Ser Gln Val Leu Gln Glu Lys Tyr Lys 500 505 510 Val Glu Leu Asp Pro Glu Tyr Thr Gln Glu Cys Asp Asp Tyr Lys Ala 515 520 525 Trp Ser Arg Asp Trp Lys Glu Cys Met Ile Arg Leu His Thr Asp Pro 530 535 540 Gln Asn His Gln Leu Ala Thr Asn Ala Ile Gly Lys Val Val Asp Pro 545 550 555 560 Gln Leu Arg Val Tyr Asp Val Lys Asn Leu Arg Val Cys Asp Ala Gly 565 570 575 Ser Met Pro Ser Pro Pro Thr Gly Asn Pro Gln Gly Ala Ile Met Val 580 585 590 Val Ala Glu Arg Cys Ala His Phe Ile Lys Gln Thr Trp Gln 595 600 605 5 583 PRT Helicoverpa zea 5 Met Ala Asp Ala Ala Ala Ser Gln Ser Ser Ala Gln Thr Val Lys Leu 1 5 10 15 Ala Leu Gln Val Leu Gln Thr Leu Ser Leu Thr Ala Trp Gln Tyr Pro 20 25 30 Pro Asp Cys Ala Leu Thr Asn Gly Ser Ser Phe Asp Phe Ile Val Val 35 40 45 Gly Ser Gly Thr Ala Gly Ser Val Leu Ala Asn Arg Leu Ser Ala Asn 50 55 60 Asp Ser Val Ser Val Leu Leu Leu Glu Ala Gly Gly Tyr Pro Pro Leu 65 70 75 80 Glu Ser Glu Leu Pro Ala Leu Phe Met Met Leu Ser Asn Ser Asp Tyr 85 90 95 Asp Tyr Lys Tyr Tyr Ala Glu Asn Asp Asn Tyr Thr Met Gln Asn Ile 100 105 110 Arg Gly Lys Arg Cys Ala Leu Thr Gln Gly Lys Val Leu Gly Gly Thr 115 120 125 Ser Ser Thr Tyr Ala Met Met His Thr Arg Gly Asp Pro Gln Asp Tyr 130 135 140 Asp Val Trp Ala Glu Arg Ala Asn Asp Thr Thr Trp Asn Ala Thr Asn 145 150 155 160 Thr Leu Ser Tyr Phe Lys Lys Gln Glu Lys Leu Thr Asp Glu Glu Leu 165 170 175 Leu His Ser Glu Tyr Ala Ala Val His Gly Thr Asp Gly Met Val Lys 180 185 190 Ile Arg Arg Glu Thr Ser Pro Leu Leu Asp Asp Ile Leu Gly Arg Ile 195 200 205 Leu Arg Gly Arg Ala Arg Phe Asn Asp Gly His His Ile Ile Glu Ser 210 215 220 Leu Arg Phe Gly Tyr Thr Gln Val Ala Ile Cys His Arg Met Met Glu 225 230 235 240 Cys Gly Gln Ser Ser Ala Leu Ala Tyr Leu Ser Ser Ala Lys Lys Arg 245 250 255 Lys Asn Leu Cys Val Ser Leu Phe Thr Thr Ala Thr Lys Ile Leu Ile 260 265 270 Glu Asn Glu Val Ala Val Gly Val Gln Leu Thr Thr Ser Thr Asn Glu 275 280 285 Thr Tyr Asn Ile Tyr Ser Asn Lys Glu Val Ile Val Ser Ala Gly Thr 290 295 300 Phe Asn Ser Pro Lys Leu Leu Met Leu Ser Gly Ile Gly Pro Arg Glu 305 310 315 320 His Leu Glu Ser Val Glu Ile Asp Val Val Ala Asp Leu Pro Val Gly 325 330 335 Gln Asn Tyr Met Asp Gln Pro Ser Ala Pro Ile Ile Ile Gln Met Asp 340 345 350 Glu Ser Ala Glu Val Ala Gly Ala Ile Asn Pro His Gln Phe Pro Leu 355 360 365 Pro Thr Phe Ile Gly Asn Val Ala Leu Asp Ser Pro Ser Lys Arg Pro 370 375 380 Gln Tyr His Thr Val Asn Phe Leu Phe Pro Ala Asn Ser Thr Asp Leu 385 390 395 400 Leu Asp Met Cys Ser Leu Phe Leu Ser Tyr Ser Asp Glu Val Cys Gln 405 410 415 Lys Val Tyr Glu Ala Thr Thr Asn Arg Lys Thr Ile Phe Ser Leu Val 420 425 430 Gly Leu Ala Leu Pro Asn Ser Arg Gly Glu Val Leu Leu Ala Ser Ala 435 440 445 Asp Pro Ala Ala Ala Pro Ile Val His Thr Gly Met Phe Ser Asn Tyr 450 455 460 Thr Asp Leu Asn Leu Met Gly Arg Ala Phe Ile Asp His Val Arg Val 465 470 475 480 Leu Asn Ser Thr Tyr Phe Arg Ser Val Asn Ala Thr Ile Leu Asp Leu 485 490 495 Gly Phe Cys Lys Asp Thr Thr Ser Glu Val Glu Phe Trp Glu Cys Tyr 500 505 510 Thr Leu Ala Met Ser Asn Thr Met Trp His Phe Gly Gly Thr Cys Ala 515 520 525 Met Gly Leu Val Leu Asp Ser Lys Met Lys Val Lys Gly Val Gly Arg 530 535 540 Leu Arg Val Val Asp Ser Ser Ser Met Pro Ala Leu Val Thr Gly Lys 545 550 555 560 Val Asn Ser Pro Ile Gly Met Leu Ala Glu Lys Ala Ala Asp Phe Ile 565 570 575 Leu Thr Asp His Asn Ile Leu 580 6 214 PRT Helicoverpa zea 6 Met Ser Ser Thr Gly Leu Thr Gln Leu Thr Gly Leu Leu Tyr Ser Ser 1 5 10 15 Tyr Ala Asp Lys Ser Arg Lys Gln Pro Asp Leu Gln Phe Phe Phe Asn 20 25 30 Gly Leu Tyr Ala Asp Cys Ser Lys Thr Gly Val Ile Gly Glu Pro Ala 35 40 45 Glu Asp Cys Ser Asp Gly Tyr Lys Ile Ser Ala Asn Ala Val Ala Leu 50 55 60 Leu Pro Arg Ser Val Gly His Val Thr Ile Asn Ser Thr Asp Pro Phe 65 70 75 80 Lys Ser Ala Leu Phe Tyr Pro Asn Phe Phe Ser His Pro Asp Asp Met 85 90 95 Asn Ile Val Met Glu Gly Val Asp Tyr Leu Arg Lys Ile Phe Glu Ser 100 105 110 Gln Val Leu Gln Glu Lys Tyr Lys Val Glu Leu Asp Pro Glu Tyr Thr 115 120 125 Gln Glu Cys Asp Asp Tyr Lys Ala Trp Ser Arg Asp Trp Lys Glu Cys 130 135 140 Met Ile Arg Leu His Thr Asp Pro Gln Asn His Gln Leu Ala Thr Asn 145 150 155 160 Ala Ile Gly Lys Val Val Asp Pro Gln Leu Arg Val Tyr Asp Val Lys 165 170 175 Asn Leu Arg Val Cys Asp Ala Gly Ser Met Pro Ser Pro Pro Thr Gly 180 185 190 Asn Pro Gln Gly Ala Ile Met Val Val Ala Glu Arg Cys Ala His Phe 195 200 205 Ile Lys Gln Thr Trp Gln 210 

What is claimed is:
 1. A polynucleotide having the sequence of SEQ ID NO: 1 and conservatively modified variants thereof.
 2. The polynucleotide of claim 1 wherein said polynucleotide is DNA.
 3. A polynucleotide having the sequence of SEQ ID NO: 2 and conservatively modified variants thereof.
 4. The polynucleotide of claim 3 wherein said polynucleotide is DNA.
 5. A polynucleotide having the sequence of SEQ ID NO: 3 and conservatively modified variants thereof.
 6. The polynucleotide of claim 5 wherein said polynucleotide is DNA.
 7. A glucose oxidase protein having the amino acid sequence of SEQ ID NO: 4 and conservatively modified variants thereof.
 8. A glucose oxidase protein having the amino acid sequence of SEQ ID NO: 5 and conservatively modified variants thereof.
 9. A glucose oxidase protein having the amino acid sequence of SEQ ID NO: 6 and conservatively modified variants thereof.
 10. A recombinant polynucleotide comprising a vector and a gene wherein said gene encodes an insect glucose oxidase protein.
 11. The polynucleotide vector of claim 9 wherein said gene is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
 3. 12. The recombinant polynucleotide of claim 9 wherein said vector is selected from the group cosisting of pGEM-T Easy, pBK-CMV, pET28a(+), pCAMBIA, pBI101, pGA482 and pRT-100.
 13. A method for producing large quantities of the polypeptide of claim 5 comprising: introducing the recombinant polynucleotide of claim 9 into host cells so as to yield transformed host cells; inducing expression the protein encoded by said polynucleotide sequence; and, extracting said polypeptide from said host cells.
 14. A method for creating a plant having enhanced resistance to insect predation comprising: introducing the recombinant polynucleotide of claim 9 into the cells of a plant so as to yield transformed plant cells; and, regenerating said transformed plant cells to provide a differentiated plant wherein said plant expresses the polypeptide encoded by said gene.
 15. The transgenic plant formed by the method of claim
 14. 16. The transgenic plant of claim 15 wherein said plant is a commercially grown crop.
 17. The transgenic plant of claim 16 wherein said plant is tobacco.
 18. A seed of the transgenic plant formed by the method of claim
 14. 19. An antibody that binds specifically to an insect glucose oxidase protein.
 20. The antibody of claim 19 wherein said glucose oxidase protein is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:
 6. 